UNIVERSITY    OF  CALIFORNIA    PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  4,  No.  12,  pp.  397-405,  3  figures  in  text  December  22,  1922 


THE  EFFECT  OF  REACTION  ON  THE  FIXATION 
OF  NITROGEN  BY  AZOTOBACTER* 

BY 

HARLAN  W.  JOHNSON  and  CHARLES  B.  LIPMAN 


INTRODUCTORY 

Azotobacter  has  been  given  considerable  attention  in  the  literature 
of  soil  bacteriology  and  it  is  thought  that  it  plays  an  important  part 
in  keeping  up  the  supply  of  nitrogen  in  many  soils. 

It  has  been  recognized,  almost  since  its  first  discovery,  that  Azoto- 
bacter is  especially  sensitive  to  the  reaction  of  the  medium  in  Avhich 
it  grows.  The  effect  of  acidity  on  the  organism  is  so  well  established 
that  its  absence  from  a  soil  has  frequently  been  taken  as  an  indica- 
tion that  the  soil  needed  lime.  In  fact,  Christensen  has  worked  out  a 
method  wherein  he  uses  these  organisms  to  determine  the  lime  require- 
ment of  soils.  The  point  has  been  repeatedly  stressed  that  the  acidity 
of  soils  must  be  neutralized  if  Azotobacter  is  to  fix  nitrogen  effectively. 

Since  the  influence  of  hydrogen-ion  concentration  on  bacteria  has 
been  recognized,  the  following  investigators  have  reported  on  its  effect 
on  these  organisms. 

Fred  and  Davenport1  reported  that  Azotobacter  is  very  sensitive, 
the  limits  for  its  growth  being  between  PH  6.6  and  PH  8.4  to  8.8. 

Gainey2  in  a  preliminary  report  states  that  in  90  soils  studied  all 
but  3  of  the  37  in  which  no  Azotobacter  was  found  had  a  PH  value  of 
5.9  or  less  and  all  but  3  of  those  containing  the  organisms  had  PH  values 
of  6.0  or  greater.  In  later  papers3  he  reported  that  Azotobacter  rapidly 
disappears  when  inoculated  into  soils  whose  PH  value  is  below  6.0  and 
that,  in  a  study  of  382  soils,  using  the  hydrogen  electrode  for  PH 
measurements,  158  samples  with  PH  values  below  6.0  and  20  above  6.0 


*  This  study  was  undertaken  at  the  suggestion  of  Dr.  C.  B.  Lipman,  in  whose 
laboratory  the  work  was  carried  out. 


398  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  i 

contained  no  Azotobacter,  while  165  samples  whose  PH  values  were 
above  6.0  and  39  samples  below  6.0  showed  Azotobacter.  The  average 
nitrogen  fixed  in  186  samples  containing  Azotobacter  was  7.9  mgs., 
while  the  average  fixed  in  181  samples  lacking  the  organism  was 
4.6  mgs. 

Waksman4  in  a  study  of  cranberry  soils  found  no  Azotobacter  in 
an  unlimed  soil  with  PH  values  of  5.4  to  5.6,  but  in  an  adjacent  limed 
soil  with  a  PH  value  of  6.2-6.4  found  it  to  be  present. 

These  investigators  agree  that  slight  acidity  inhibits  the  growth 
of  Azotobacter.  None  of  them,  however,  reports  on  the  effect  of 
the  reaction  on  the  nitrogen  fixing  efficiency  of  the  organism.  Gainey 
reports  the  nitrogen  fixed  in  the  soils  containing  Azotobacter  as  com- 
pared with  soils  lacking  them,  but  does  not  state  the  effect  of  the 
reaction  in  the  soils  where  they  were  present. 

The  work  here  presented  was  undertaken  to  determine  the  effect  of 
various  hydrogen-ion  concentrations  on  the  ability  of  Azotobacfi  r 
chroococcum  to  fix  nitrogen. 

EXPEEIMENTAL 

The  organism  used  was 'a  strain  of  Azotobacter  chroococcum 
isolated  from  a  California  soil  which  in  previous  work  had  been  found 
to  be  very  efficient  in  nitrogen  fixation  and  to  produce  abundant 
pigment. 

The  medium  was  made  up  as  follows: 

Mannite,  15.0  gms. 

MgS04  •  7H20,       .2  gms. 
NaCl,  .2  gms. 

CaSO,  •  2H.O,        .1  gms. 
H3P04,  1.5  c.c. 

Distilled  water,  1000  c.c. 

This  solution  was  titrated  with  N/2  KOH  to  give  definite  PH  values, 
using  the  hydrogen  electrode  in  the  titration.  The  titration  curve  of 
the  medium  is  shown  in  figure  1.  The  inoculations  were  made  into 
100  c.c.  portions  of  the  medium  in  800  c.c.  Erlenmeyer  flasks.  To 
secure  uniform  inoculation,  50  c.c.  portions  of  the  medium  in  a  small 
Erlenmeyer  flask  were  inoculated  with  Azotobacter,  and  after  a  char- 
acteristic membrane  had  formed  the  flask  was  shaken  vigorously,  and 
(he  suspension  was  used  as  the  inoculum. 


^»        Cn 


Ph  Values 


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Kt 

<4 

6 
S 

5 

c 

\ 

o 

ft) 

\ 

"^1 

L-  . 

0 

0 

<5> 

v. 

Co 

* 

*> 

m 

o 

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I 

400  University  of  California  Publications  in  Agricultural  Sciences       [Vol.4 

The  First  Series 

In  this  series  the  medium  was  titrated  to  give  PH  values  of  approxi- 
mately 3,  4,  5,  6,  7,  8,  9,  10,  and  11.  These  values  changed  during 
sterilization  and  incubation. 

Four  flasks  of  each  reaction  were  inoculated  and  incubated  three 
weeks  at  28°  C. 

Three  of  the  flasks  of  each  reaction  were  left  undisturbed.  The 
others  were  used  for  PH  measurements,  5  c.c.  being  withdrawn  at 
intervals  from  each  for  electrometric  determinations. 

The  PH  values  of  the  solutions  in  these  flasks  at  the  time  of 
inoculation  and  at  each  of  the  succeeding  periods  are  shown  in  figure  2. 

After  incubating  twenty  days  the  nitrogen  in  each  of  the  un- 
disturbed flasks  was  determined  and  the  amounts  fixed  are  given  in 
table  1. 

TABLE  1 

Nitrogen  Fixed  in  Solutions  op  Various  Ph  Values 

Series  1 

N  Fixed  Mgs. 


Ph  at  time 

Ph  at  time 

Flask 

Flask 

Flask 

of  titration 

of  inoculation 

No.  1 

No.  2 

No.  3 

Averag 

3.14 

3.47 

.07 

.0 

.0 

.02 

4.10 

4.80 

.0 

.07 

.0 

.02 

5.06 

5.31 

.0 

.0 

.0 

.00 

5.98 

6.07 

.70 

1.12 

.0 

.61 

7.00 

7.08 

1.82 

2.66 

2.52 

2.33 

8.15 

7.69 

2.59 

1.75 

2.45 

2.26 

9.01 

7.98 

2.42 

2.28 

2.42 

2.37 

10.04 

8.08 

2.40 

3.38 

3.52 

3.10 

11.03 

8.86 

4.95 

4.11 

3.41 

4.16 

It  will  be  noticed  that  the  PH  values  of  the  media  were  not  the 
same  when  inoculated,  as  at  the  time  of  titration.  This  may  be  due 
to  incomplete  reaction  at  the  time  of  titration,  although  the  titration 
required  two  days  for  completion.  It  might  be  due  to  the  absorption 
of  carbon  dioxide  by  the  more  alkaline  media,  since  these  media  showed 
the  greatest  changes  and  several  days  intervened  between  sterilization 
and  inoculation.  Probably  both  factors  had  their  effect.  It  will  be 
noted  that  the  solutions  with  PH  values  of  6.0  and  7.0,  whose  reactions 
remained  remarkably  constant,  lie  in  the  region  which  is  shown  by 


1922]  Johnson-Lipman :  The  Fixation  of  Nitrogen  by  Azotobacter 


401 


the  curve  in  figure  1  to  be  most  highly  buffered,  while  those  with  PH 
values  between  8.0  and  11.0,  which  are  in  a  poorly  buffered  region, 
show  the  greatest  changes.  The  growth  of  Azotobacter  in  the  more 
alkaline  solutions  also  affects  the  reaction.  The  more  acid  solutions, 
although  in  a  poorly  buffered  region,  show  little  change  in  reaction. 


Fig.  2.     Changes  in  Ph  Values.     First  Series 


II    -t.16 
6-      .61 


S~  00 


4  —  O.Z 


-3 


0.2. 


i/„  J     a.     J     *     S     6     7 


fff    Jilrsfiui 


8     9      ID     II     13.    13     if    /S    16    17     is    /9    zo 

Days  after  Inoculation 


The  end  point  for  nitrogen  fixation  on  the  acid  side  of  the  neutral 
point  evidently  lies  very  close  to  PH  6.0.  At  that  reaction  only  a  very 
small  amount  of  nitrogen  was  fixed  and  in  the  solutions  with  lower 
PH  values  no  fixation  occurred.  The  alkaline  reaction  inhibiting 
fixation  was  not  reached  in  this  series;  in  fact  the  largest  amount  of 
N  was  fixed  in  the  most  alkaline  medium  used. 


402  University  of  California  Publications  in  Agricultural  Sciences       [Vol.4 

The  Second  Series 

The  second  series  was  planned  to  find  more  accurately  the  lower 
critical  PH  value  and  an  alkaline  reaction  inhibiting  fixation.  Conse- 
quently solutions  were  made  up  with  PH  values  of  approximately  5,  6. 
6.2,  6.4,  6.6,  6.8,  7,  8,  9,  10,  11,  and  12. 

Four  flasks  of  each  PH  value  were  again  inoculated  and  incubated, 
one  of  each  PH  value  being  used  as  before  for  PH  determinations. 

Only  three  PH  determinations  were  made,  viz.,  at  inoculation,  ten 
days,  and  seventeen  days  later.  It  is  regretted  that  no  determination 
was  made  at  the  end  of  incubation.  The  reactions  at  the  time  of 
inoculation  and  later  are  shown  in  figure  3.  Again  those  in  the 
buffered  region  between  PH  6.0  and  7.0  showed  little  change,  while 
those  above  in  the  less  buffered  region  were  markedly  changed.  The 
change  in  the  highly  buffered  solution  of  PH  12  is  remarkable,  but  must 
most  likely  be  due  to  absorption  of  carbon  dioxide  since  sterilization  did 
not  materially  change  the  reaction.  It  should  be  noted  that  an  extra 
flask,  uninoculated,  changed  exactly  the  same  as  the  inoculated  one. 

These  cultures  were  incubated  thirty  days,  since  the  amounts  of 
nitrogen  fixed  in  the  period  of  twenty  days  in  the  first  series  were 
rather  small.  The  amounts  of  nitrogen  fixed  in  this  series  are  given 
in  table  2. 

TABLE  2 

Nitrogen  Fixed  in  Solutions  of  Various  Ph  Values 

Series  2 

N  Fixed  Mgs. 


Ph  at  time 

Pn  at  time 

Flask 

of  titration 

of  inoculation 

No.  1 

5.00 

5.34 

.0 

5.99 

5.97 

.28 

6.20 

6.29 

5.88 

(5.41 

6.49 

3.36 

6.63 

6.70 

3.64 

6.83 

6.83 

3.78 

6.97 

6.91 

7.50 

8.00 

7.93 

5.46 

8.99 

8.18 

5.46 

10.02 

8.52 

5.60 

11.02 

9.26 

.14 

12.05 

11.87 

.0 

Flask 

Flask 

No.  2 

No.  3 

Averaga 

.0 

.28 

.09 

.84 

1.96 

1.02 

4.70 

4.20 

4.95 

4.62 

5.18 

4.38 

4.90 

1.34 

4.28 

4.90 

4.34 

4.34 

8.12 

2.66 

7. SI 

5.18 

4.48 

r..04 

4.90 

5.34 

5.23 

5.88 

.42  T 

3.97 

8.26* 

.0 

.07 

.0 

.14 

.05 

■iitly  some 

factor  hai 

l  affected  tlio 

*  Not  included  in  averages  since  very  < 
results. 

The  amounts  of  nitrogen  fixed  in  this  series  were  somewhat  larger 
than  those  in  the  first  series  and  show  a  somewhat  different  effect  of 

reaction.     In  this  series  the  neutral  solution  shows  the  greatest  fixation. 


1922] 


Johnson-Lip  man:  The  Fixation  of  Nitrogen  by  Asotobaeter 


403 


It  is  evident  again  that  PH  6.0  is  near  the  critical  limit  for  fixation. 
The  very  great  increase  in  N  fixed  in  the  solution  about  .2  PH  higher 
is  remarkable.    The  lower  limit  for  fixation  is  apparently  very  definite. 

The  upper  limit  for  nitrogen  fixation  was  reached  in  the  solution 
whose  PH  value  at  inoculation  was  9.26. 


Fig.  3.     Changes  in  Ph  Values.     Second  Series 


s  a 


s 

fff  irtoculaTtan 
flf-   hfrafton 


Days  after  Inoculation 


404  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  4 


GENERAL  DISCUSSION 

These  experiments  -were  planned  to  determine  the  effects  of 
hydrogen-ion  concentration  on  nitrogen  fixation  by  Azotobacter.  The 
results  obtained,  however,  can  only  be  used  to  show  that  nitrogen 
fixation  is  not  seriously  affected  until  the  critical  limits  of  reaction  are 
closely  approached,  when  an  abrupt  decrease  occurs  in  the  amount  of 
the  nitrogen  fixed.  The  variations  in  amounts  of  nitrogen  fixed  in 
solutions  between  these  limits  are  not  sufficient,  considering  the 
number  of  cultures  used  and  the  variations  between  cultures  of  the 
same  reaction,  to  be  of  definite  significance.  The  averages  given  in  the 
tables  are  simply  for  convenience,  and  it  is  realized  that  a  large 
number  of  flasks  of  each  reaction  would  be  necessary  in  order  to 
secure  conclusive  evidence  of  the  effects  of  various  reactions. 

The  results  show  that  there  is  an  abrupt  decrease  in  the  amount 
of  nitrogen  fixed  between  PH  6.2  and  PH  6.0,  in  other  words,  that  the 
limiting  hydrogen-ion  concentration  for  good  nitrogen  fixation  is  a 
definite  value  between  those  two  points.  This  corroborates  the  results 
of  the  previous  investigators  who  found  Azotobacter  in  soils  whose 
PH  was  6.0  or  above  and  none  in  soils  with  lower  PH  values.  It  shows 
that  we  can  expect  no  nitrogen  fixation  by  Azotobacter  in  many  of  our 
soils,  since  it  has  repeatedly  been  shown  that  PH  values  below  6.0  are 
frequently  encountered.  These  organisms  apparently  react  much  more 
sensitively  than  do  most  of  our  other  soil  organisms,  since  ammoni- 
fication,  nitrification,  and  other  forms  of  bacterial  activity  are  active 
in  soils  whose  acidity  is  higher  than  PH  6. 

The  alkaline  limit  for  nitrogen  fixation  is  apparently  near  PH  9.0, 
since  in  the  first  series  the  solution  whose  PH  value  was  8.86  at  inocu- 
Lation  showed  good  fixation,  while  in  the  second  series  the  solution 
inoculated  at  PH  9-26  showed  no  nitrogen  fixed.  It  is  doubtful 
whether  many  soils  ever  attain  such  a  reaction.  Sharp  and  Hoagland 
report  two  soils  whose  alkalinity  is  greater,  but  these  were  exceptional 
alkali  soils. 

From  this  study,  Fred's1  limits  of  PH  6.6  and  S.4  to  8.8  would 
seem  to  be  too  narrow.  Different  strains  of  Azotobacter,  however,  may 
show  variations  in  the  effect  of  reactions  on  growth,  and  since  this 
strain  was  an  especially  vigorous  one  its  limits  might  be  expected  to  be 
wider. 


1922]  Johnson-Lipman :  The  Fixation  of  Nitrogen  by  Azotobacter  40f 


SUMMARY 

A  vigorous  strain  of  Azotobacter  chroococcum  was  grown  in 
solutions  whose  reactions  were  definitely  determined  by  the  hydrogen 
electrode.  The  nitrogen  fixed  in  the  solutions  of  each  reaction  was 
determined  and  the  changes  in  reaction  during  incubation  were 
measured. 

It  was  found  that  the  reaction  of  the  solutions  below  PH  8.0 
changed  very  little,  those  below  PH  6.0  because  no  growth  occurred, 
and  those  between  6.0  and  8.0  because  the  solution  in  this  region  was 
highly  buffered. 

Above  PH  8.0  the  reaction  changed  greatly,  possibly  due  to  incom- 
plete reaction  of  the  alkali  at  the  time  of  titration,  but  more  probably 
due  to  absorption  of  carbon  dioxide  by  the  strong  alkali. 

The  amount  of  nitrogen  fixed  was  not  greatly  affected  by  reactions 
between  PH  values  of  6.2  and  8.8  although  reactions  around  PH  7.0 
and  8.0  seemed  to  be  most  favorable.  Slight  changes  outside  of  these 
values  caused  an  abrupt  decrease  in  fixation. 


LITERATURE  CITED 

i  Fred,  E.  B.,  and  Davenport,  A. 

1918.     Influence  of  reaction  on  nitrogen  assimilating  bacteria.    Jour.  Agr.  Res., 
vol.  14,  pp.  317-336. 

2  Gainet,  P.  L. 

1918.     Soil  reaction  and  the  growth  of  Azotobacter.    Jour.  Agr.  Res.,  vol.  14, 
pp.  265-271. 

a  Gainey,  P.  L. 

1922a.  A  study  of  some  factors  controlling  the  presence  of  Azotobacter. 
1922b.  Correlation  between  the  presence   of  Azotobacter  in  a   soil  and  the 
hydrogen-ion  concentration  of  the  soil.     Scientific  Proceedings  of 
the  Society  of  American  Bacteriologists,  abstracts  32  and  33,  in 
Abst.  of  Bact.,  vol.  6,  pp.  14-15. 
*  Waksman,  S.  A. 

1918.     The  occurrence  of  Azotobacter  in  cranberry  soils.     Science,  vol.  48, 
p.  653. 


